Since its discovery by Sutherland (Ann. Rev. Biochem., 37, 149 (1968)) the central role of cyclic adenosine-3',5'-monophosphate (cAMP) as a cellular signal transduction molecule (second messenger) has been validated in numerous biochemical processes.
The corresponding guanosine-3',5'-monophosphate (cGMP) obviously controls also quite a lot of cellular biological functions, e. g. the relaxation of contracted muscle of the aorta or the inhibition of thrombocyte function (Walter, U., Rev. Physiol. Biochem. Pharmacol., 113, 42-88 (1989)).
cGMP can either support the cAMP signal pathway or can stimulate a contrary effect as a regulatory opponent.
Normally, cAMP and cGMP are formed by intracellular cyclases from ATP and GTP, respectively, after external stimulation by a hormone. During the signal transduction process both compounds bind to specific protein kinases which in turn can activate or deactivate target proteins by phosphorylation.
Up to now several different cyclic nucleotide-dependent phosphodiesterases are known which metabolize cAMP and cGMP (Beavo, J. A., Second Messenger and Phosphoprotein Res., 22, 1-38 (1988)) and which are responsible for switching off a given signal.
Since several cellular functions are connected with unnaturally elevated or depressed low levels of cAMP oder cGMP, there is considerable interest in basic research on corresponding second messenger systems mainly on the level of suitable modeling cell culture experiments.
In cultured cells intracellular levels of cAMP or cGMP which are too low, for example, can be compensated by specific inhibitors of cyclic nucleotide-dependent phosphodiesterases (Nicholson et al., TIBS 12, 19-27 (1991)) preventing metabolic degradation and resulting in cyclic nucleotide accumulation.
In addition, application of membrane permeable chemical modifications of cAMP and cGMP such as 8-bromo-cAMP/cGMP or 8-(4-chlorophenyl-thio)-cAMP/cGMP can directly activate the corresponding protein kinases in order to compensate low cyclic nucleotide levels. Chemically modified cyclic nucleotides can also be used for the in vitro characterization of kinase specificity (Wolfe et al. J. Biol. Chem., 264, 7734-7741 (1989), Geiger et al., Proc. Natl. Acad. Sci. USA, 89, 1031-1035 (1992)).
Finally, use of cyclase stimulators forskolin, cholera toxin, nitroprussid, NO) results in higher production rates of the corresponding second messenger, i.e. the intracellular concentration of cAMP and cGMP can be increased.
On the other hand, a potential decrease or even a complete inhibition of the cAMP or cGMP signal pathway is of predominent importance as well.
Presently, some isoquinoline derivatives (Chijawa et al., J. Biol. Chem., 265, 5267 (1990)) and a protein-based structure (Walsh et al., J. Biol. Chem., 246, 1977, (1971)), are used as inhibitors of cyclic nucleotide-dependent protein kinases. In addition, R.sub.P -configurated derivatives of adenosine-3',5'-monophosphorothioate (R.sub.P -cAMPS) have been used for inhibition of cAMP-mediated biological effects.
R.sub.P -cAMPS is an antagonist of cAMP and a competitive inhibitor of cAMP-dependent protein kinases type I and II (cAK I & II) (Botelho et al., Methods Enzymol., 159, 159 (1988)). Other diastereomeric cAMPS derivatives with R.sub.P -configuration were already synthesized (Dostmann et al., J. Biol. Chem., 265, 10484 (1990)). Their biological properties are presently tested.
With respect to cAMPS derivatives it has been found that not all R.sub.P -configurated diastereomers necessarily must be inhibitors of cAK but also can have agonistic effects (Sandberg et al., Biochem. J., 279, 521-527 (1991)). Most of the about 20 R.sub.P -isomers tested so far are only partial antagonists or rather agonists of cAK and up to now there is no structural criterion which allows a reliable prediction whether or not a certain R.sub.P -configurated adenosine-3',5'-monophosphorothioate derivative can completely block cAK I or cAK II, respectively.
In contrast to cAMPS derivatives only two sulfur-modified compounds of cGMP with inhibitory properties have been described: R.sub.P -Guanosine-3',5'-monophosphorothioate (R.sub.P -cGMPS) and R.sub.P -8-chloroguanosine-3',5'-monophosphorothioate (R.sub.P -8-Cl-cGMPS) (Butt et al., FEBS Lett., 263, 47 (1990)).
However, a potential use of these compounds as inhibitors of the cGMP signal pathway is opposed by severe drawbacks. Particularly, R.sub.P -cGMPS lacks the required specificity for cGMP-dependent protein kinase (cGK) and inhibits cAK as well. In addition, the required lipophilicity which is important for cell membrane penetration properties is rather poor for both compounds, although hydrophobicity in average is doubled by modification with sulfur (Braumann et al., J. Chromatogr., 350, 105-118 (1985)).
Therefore, the object of the invention is to find new derivatives of cGMP which exhibit considerably higher lipophilicity and cell membrane permeability and which can inhibit cGK competitively and selectively. In addition such compounds should have sufficient chemical, physical and metabolical stability which would enable their use as stable ligands for affinity chromatography.